Hybrid SID, IM, and UVPD Methods for Complex-Down MS of Protein Complexes

为了实现它们的功能，许多蛋白质复合物必须首先与多个其它蛋白质复合物形成动态复合物。 蛋白质或结合伴侣。总体化学计量、拓扑结构以及亚基间和亚基内的表征 蛋白质和核蛋白复合物的接触以及它们的组装/拆卸是至关重要的，因为这些 复合物调节关键的生物过程。天然质谱法（nMS），特别是与 离子迁移率（IM）和活化方法，例如碰撞诱导解离（CID）、紫外光活化和离子迁移率（IM）。 光解离（UVPD）和表面诱导解离（SID）正在成为一种强大的技术， 这对研究这些复杂系统和指导其他结构生物学工具的适当应用具有重要意义。 尽管nMS对结构生物学有着巨大的潜力，但商业仪器缺乏许多必要的工具， 充分表征这些复合物。SID尚未商业化，但已被证明是一种非常有用的 蛋白质复合物研究中的工具，切割复合物中最弱的界面并产生亚- 反映结构连接性的复合物。虽然IM在一些商业上是可用的， 平台，分辨率往往不足以进行详细的结构研究。在TR&D1中，我们建议 执行更高能量SID，在多个不同仪器上进行更有效的碎片离子收集 平台在TR&D2中，我们建议在高分辨率Orbitrap仪器上开发高分辨率IM。在这 TR&D我们建议将TR & D 1和2中开发的技术与供应商原型IM结合起来 设备，以便能够使用集成的，高效的工作流程对蛋白质复合物进行全面表征。 SID和IM的耦合是必不可少的，因为当SID置于IM之前时，它允许构象信息 即使当峰重叠时，也可以在完整复合物和由SID产生的亚复合物上获得 m/z空间，从而能够构建结构模型。当SID放置在IM之后时，它允许不同的构象 完整的复合物（如果存在），以单独的迁移率选择碎片。除了耦合SID 和IM，我们建议将这些方法与UVPD结合联合收割机。这允许对复杂的 组装和与SID和IM的亚基连接，以及共价片段化（肽的测序 主链）。这种方法将有利于辨别配体结合位点沿着配体结合位点。 任何翻译后修饰（PTM）。我们建议在多个工具平台上进行此操作，包括 沃茨Synapt G2（S）、Thermo（Q）Exactive和Bruker FTICR。多平台的使用是必要的 因为每个平台具有不同的质量分辨率、灵敏度和速度，并且某些平台将更适合 某些复杂的。因此，与多个平台的结合允许定制实验， 利益的复杂性。使用多种平台也是必不可少的，因为它可以更好地传播到 更广泛的社区，他们可能只能访问其中一个平台。

In order to fulfill their functions many protein complexes must first form dynamic complexes with multiple other proteins or binding partners. Characterization of the overall stoichiometry, topology, and inter- and intra-subunit contacts of protein and nucleoprotein complexes, and their assembly/disassembly, is critical because these complexes regulate key biological processes. Native mass spectrometry (nMS), particularly in combination with ion mobility (IM), and activation methods such as collision-induced dissociation (CID), ultraviolet photodissociation (UVPD), and surface induced dissociation (SID), is emerging as a powerful technique with which to study these complex systems and for guiding appropriate application of other structural biology tools. Despite the promise of nMS for structural biology, commercial instruments lack many of the tools necessary to fully characterize these complexes. SID, which is not yet commercialized, has proven to be an incredibly useful tool in the study of protein complexes, cleaving the weakest interfaces in the complex and producing sub- complexes that are reflective of the structure’s connectivity. While IM is commercially available on some platforms, the resolution is often insufficient for detailed structural studies. In TR&D1, we propose to enable higher energy SID to be performed, with more efficient fragment ion collection on multiple different instrument platforms. In TR&D2 we propose to develop high-resolution IM on a high-resolution Orbitrap instrument. In this TR&D we propose to couple the technologies developed in TR&Ds1 and 2, in addition to vendor prototype IM devices, in order to enable the full characterization of protein complexes using integrated, efficient workflows. Coupling of SID and IM is essential because when SID is placed before IM it allows conformational information to be obtained on the intact complex and the subcomplexes produced from SID even when the peaks overlap in m/z space, enabling structural models to be built. When SID is placed after IM, it allows different conformations of the intact complex (if present) to be individually mobility-selected for fragmentation. In addition to coupling SID and IM, we propose to combine these approaches with UVPD. This allows for the interrogation of complex assembly and subunit connectivity with SID and IM, with covalent fragmentation (sequencing of the peptide backbone) from UVPD. This approach will be beneficial in discerning ligand binding sites along with the sites of any post-translational modifications (PTMs). We propose to do this on multiple instrumental platforms, including the Waters Synapt G2(S), Thermo (Q) Exactive, and Bruker FTICR. The use of multiple platforms is necessary as each platform has different mass resolution, sensitivity, and speed, and certain platforms will be better suited to certain complexes. Hence incorporation with multiple platforms allows the experiments to be customized to the complex of interest. The use of multiple platforms is also essential as it allows better dissemination to the wider community, who may have access to only one of these platforms.